The Bordetella genus includes several closely related subspecies of Gram-negative bacteria that colonize ciliated respiratory epithelial surfaces in mammals. In addition to their importance as infectious agents, members of the Bordetella genus provide excellent models for probing bacterial-host interactions. The availability of highly related subspecies with different host adaptations allows comparative studies of pathogenesis. Although B. pertussis is exclusively adapted to humans, B. bronchiseptica has a remarkably broad host range that includes a variety of laboratory animals. B. bronchiseptica models allow assessments of the molecular basis of pathogenesis in the context of natural host-parasite interactions. We have identified a type III secretion system (TTSS) in B. bronchiseptica that appears to play a key role in facilitating persistent infection of the respiratory epithelium. Recent results indicate that type III secretion (TTS) apparatus genes, regulatory genes, and genes encoding secreted proteins are actively transcribed in B. pertussis. Furthermore, the BtrS regulatory system that controls TTS is present and functional. The objective of this proposal is to conduct a comprehensive comparative analysis of TTS in Bordetella subspecies infectious for humans and other animals. Results from our studies will contribute to a fundamental understanding of mechanisms of pathogenesis and the evolution of bacterial virulence. Specifically, we propose to: 1. Conduct a comparative analysis of the BtrS regulons in Bordetella subspecies. BtrS is a newly identified sigma factor that sits at the top of a complex regulatory hierarchy controlling expression of type III secretion loci and other genes. These studies have the potential to discover novel virulence factors and regulatory mechanisms. 2. Determine the roles of "partner switcher" homologs in the regulation of type III secretion. Partner switching represents a new and expanding paradigm in bacterial regulation. We will test the hypothesis that differences in the behavior of the BtrU,V,W partner switching complex accounts for differences in the control of type III secretion between Bordetella subspecies. 3. Investigate the effects of type III secretion and other BtrS-regulated phenotypes during respiratory tract infection by B. pertussis and B. bronchiseptica.